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Abstract Abstract 1711 Background: Mutations in genes of the splicing machinery, such as SF3B1, SRSF2 and U2AF35 are common in patients with myelodysplastic syndromes [MDS] (Nature 2011;478:64) and chronic myelomonocytic leukemia [CMML] (Haematologica 2012;Epub). In MDS, SRSF2 gene mutations are an independent risk factor for shortened over-all (OS) and leukemia-free survival (LFS) (Blood 2012;119:3578). In MDS with ring sideroblasts (RS), SF3B1 mutations have a high prevalence (∼50%), but do not influence either, the OS or the LFS (Blood 2012;119:569). We carried out this study to evaluate the prevalence, clinical correlates and prognosis of the aforementioned spliceosome mutations in CMML. Methods: The study included 227 patients with WHO defined CMML who were seen at the Mayo Clinic from 1997 through 2007. All patients underwent bone marrow (BM) examination and cytogenetic evaluation at diagnosis. DNA was interrogated in the three most frequent spliceosome genes with somatic mutations; SRSF2, SF3B1 and U2AF35. Results I: Prevalence and clinical correlates Among the 227 study patients, 153 (67%) were male, median age was 71 years (range, 17–90 years) and 192 (85%) met the WHO criteria for CMML-1. Ninety (40%) patients had SRSF2 mutations (86% CMML-1), 13 (6%) had SF3B1 mutations (75% CMML-1) and 20 (9%) had U2AF35 mutations (95% CMML-1). One-hundred and twenty three (54%) patients had at least one of three spliceosome mutations (86% CMML-1). Mutational hot spots were P95 for SRSF2 (P95L-n=36/H-n=32/R-n=13/A-n=1), K700E (n=7) and H662Q (n=2) for SF3B1, and Q157 (Q157R-n=5/P-n=5/G-n=1) and S34F (n=7) for U2AF35. Seven patients (54%) with SF3B1 mutations had ≥1% RS, with 5 (38%) showing ≥15% RS. Mutations involving all three spliceosome genes were mutually exclusive. The cytogenetic distribution based on the Spanish risk stratification system (Haematologica 2011;96:375) was; SRSF2 mutations: 69 (77%) low risk, 11 (12%) intermediate risk, and 10 (11%) high risk (+8-n=3, del/monosomy 7-n=2, monosomal karyotype-n=5); SF3B1 mutations: 8 (62%) low risk and 5 (38%) intermediate risk; U2AF35 mutations: 15 (75%) low risk, 3 (15%) intermediate risk and 2 (10%) high risk (p=0.89). The distribution of mutations according to the MD Anderson prognostic scoring system [MDAPS] (Blood 2002;99:840) was; SRSF2 - low-n=41, intermediate-1-n=26, intermediate-2-n=18, high-n=5, SF3B1- low-n=7, intermediate-1-n=3, intermediate-2-n=2, high-n=1, and U2AF35- low-n=11, intermediate-1-n=5, intermediate-2-n=3, high-n=1 (p=0.73). There was no statistically significant difference, among the three mutation groups, in prognostically relevant parameters, including gender distribution, median age, hemoglobin values, platelet counts, peripheral blood (PB) and BM blast counts, absolute neutrophil counts (ANC) and absolute monocyte counts (AMC). The only notable difference was that patients with the SF3B1 mutation had a lower median white blood cell count (p=0.04) and a lower absolute lymphocyte count (p=0.045). Results II: Prognostic impact of spliceosome mutations At a median follow-up of 15 months, 166 (73%) deaths and 33 (14.5%) leukemic transformations were documented. Median survivals for patients with mutations involving SRSF2, SF3B1 and U2AF35 were 24, 17 and 12 months, respectively. In univariate analysis, the presence of SRSF2 (p=0.67), SF3B1 (p=0.96) or U2AF35 (p=0.49) mutations had no prognostic impact on OS. Similarly, none of the three spliceosome mutations affected LFS; corresponding p values were 0.55 for SRSF2, 0.9 for SF3B1 and 0.38 for U2AF35 mutations respectively. We then examined possible prognostic value of having none of these mutations (n=104) vs otherwise (n=123) and the results were once again negative (p=0.87). Conclusions: SRSF2 is the most frequently mutated spliceosome gene in CMML, but neither it nor SF3B1 or U2AF35 mutations affect overall or leukemia-free survival in CMML. Furthermore, the current study suggests limited genotype-phenotype association, save for the already established association between SF3B1 mutations and RS. Disclosures: No relevant conflicts of interest to declare.

Abstract Abstract 1742 Background Karyotype is one of the most potent and reproducible risk factors for both overall (OS) and leukemia-free (LFS) survival in primary myelofibrosis (PMF) (Blood 2011;118:4595). It is currently not clear if the number of metaphases studied or the abnormal metaphase percentage alters this prognostic impact. Methods: An updated Mayo Clinic database of karyotypically- and DIPSS-plus-annotated patients with PMF was used to identify a consecutive series of patients and their cytogenetic information obtained at time of referral was centrally re-reviewed. Cytogenetic results were interpreted and reported according to the International System for Human Cytogenetic Nomenclature; abnormal karyotype was defined by the presence of at least 2 metaphases with structural abnormalities or monosomy or 3 metaphases with polysomy, regardless of number of metaphases examined. For this particular study, the presence of less than 20 evaluable metaphases did not disqualify patients. “Very high risk” karyotype included monosomal karyotype, inv(3) or i(17q) abnormalities (Blood 2011;118:4595). “unfavorable” karyotype included complex or any sole or two abnormalities that included +8, −7/7q-, -5/5q-, inv(3), i(17q), 12p-, or 11q23 rearrangement (Blood 2011;118:4595). All other cytogenetic abnormalities were considered “favorable” Results: A total of 590 patients (median age 65 years; range 19–89 years) including 424 (72%) males. The DIPSS-plus (JCO 2011;29:392) risk distribution was 40% high, 39% intermediate-2, 12% intermediate-1 and 9% low. Cytogenetic findings included 17 (3%) very high risk, 69 (12%) unfavorable, 165 (28%) favorable and 339 (57%) normal karyotypes. The number of bone marrow metaphases studied to report these cytogenetic findings were ≥20 in 468 (79%) patients, 11 to 19 in 71 (12%) patients and ≤10 in 51 (9%) patients; the proportion of cases studied with ≥20 metaphases were 53% for very high risk, 74% for unfavorable, 83% for favorable and 80% for normal karyotype (p=0.006). Among patients with abnormal karyotype, the abnormal metaphase percentage was ≥75% in 148 (59%) patients, 50 to 74% in 36 (15%) patients, 26 to 49% in 27 (11%) patients and ≤25% in 38 (15%) patients; the proportion of patients with ≥75% was 59% for very high risk, 67% for unfavorable and 56% for favorable karyotypes (p=0.70). As expected, OS was significantly different among very high risk, unfavorable, favorable and normal karyotype patients with respective median survivals of 8, 23, 41 and 57 months (p<0.0001). The number of metaphases studied (p=0.62) or the abnormal metaphase percentage (p=0.12), by themselves, did not affect survival. Similarly, the survival difference among the aforementioned cytogenetic risk groups was equally apparent when patients with ≥20 metaphases studied (n=468; P<0.0001) and those with <20 metaphases studied (n=122; p<0.0001) were separately analyzed. Analysis of patients with very high risk or unfavorable karyotype (n=86) revealed no significant effect of abnormal metaphase percentage on survival (Figure; p=0.80). A similar scenario was demonstrated for patients with favorable karyotype (Figure; p=0.24). Conclusions: Neither the number of metaphases examined nor the abnormal metaphase percentage appear to influence the currently recognized cytogenetic risk stratification in PMF. The current study has implications for both clinical practice and clinical research involving cytogenetic prognostication in hematological malignancies. Disclosures: No relevant conflicts of interest to declare.

Abstract Abstract 2902 Background: Although chronic lymphocytic leukemia (CLL) is a low grade B-cell malignancy, 2–8% of patients (pts) are reported to undergo transformation to diffuse large B-cell lymphoma (DLBCL; Richter's syndrome [RS]). Whether the risk of transformation is related to the underlying biology of CLL or is a consequence of treatment for CLL is uncertain. Also, the role of prognostic markers such as immunoglobulin heavy chain (IGHV) gene mutation status, ZAP-70 and CD38 expression, and genetic abnormalities detected by fluorescence in situ hybridization (FISH) in predicting the development of RS is not well defined. The available data on stem cell transplant (SCT) in pts with RS is based on retrospective case series of selected RS pts and is of uncertain relevance to the majority of CLL pts experiencing RS. Therefore, we conducted a cohort study of newly diagnosed CLL pts to explore the incidence and natural history of RS. Methods: The Mayo Clinic CLL database includes all consenting pts with a pathologic diagnosis of CLL. Information on date of diagnosis, treatment history, and disease-related complications is abstracted from clinical records on all pts and then maintained prospectively. We used this database to identify newly diagnosed pts with CLL from January 2000 through July 2011, who were seen at our institution within 12 months of diagnosis. Pts with biopsy-confirmed RS were included in this analysis. For pts referred from an outside institution, pathology review at Mayo Clinic was mandatory to be included in this study. The Mayo Clinic Institutional Review Board approved the study. Results: Between January 2000 and July 2011, 1641 pts with newly diagnosed CLL were seen at Mayo Clinic within 12 months of diagnosis, and this cohort had a median follow-up of 4.0 yrs (range, 0–12.3 yrs). Of these pts, 37 (2.3%) were diagnosed with RS. The median time to development of RS after a diagnosis of CLL was 1.8 yrs (range, 0–11.7 yrs). The incidence of RS at 2 and 5 yrs post CLL diagnosis were 1.3% and 2.1% respectively (Figure). Pts who developed RS had more advanced (Rai III/IV) stage disease (22% vs. 6%; p<0.001) and higher beta 2-microglobulin levels (median 3.1 mg/dL vs. 2.3 mg/dL; p=0.003) at diagnosis of their CLL compared to pts without RS. Pts who developed RS were also more likely to be positive for ZAP-70 (58% vs. 33%; p=0.03), CD49d (77% vs. 31%; p=0.0004), and CD38 (57% vs. 29%; p=0.001), have unmutated IGHV (76% vs. 44%; p=0.003) and have either del(11q22.3) or del(17p13) on FISH analysis (46% vs. 13%; p<0.0001). RS transformation occurred prior to treatment of CLL in 17 (46%) pts and after treatment of CLL in 20 pts (54%). Treatment for CLL was associated with an increased risk of RS (92% vs 34%; p<0.0001). The odds ratio (OR) of RS was increased for pts who received purine nucleoside analogues (OR=3.0; 95%CI=[1.6, 5.9]; p=0.001), alkylating agents (OR=2.5; 95% CI=[1.3, 4.9]; p=0.004), or both (OR=3.3; 95% CI=[1.7, 6.4]; p=0.0003) for treatment of their CLL. Median overall survival after development of RS was 2.1 yrs. Most pts (29/37, 78%) received R-CHOP (rituximab, cyclophosphamide, doxorubicin, vincristine and prednisone) therapy. Two (5%) pts underwent an autologous SCT and 5 (14%) underwent an allogeneic SCT. Additionally, 1 (3%) patient received an autologous SCT followed by an allogeneic SCT. By the end of the study period, 17 (46%) pts with RS had died. Conclusion: The overall rate of biopsy-proven RS in this large cohort of pts with newly diagnosed CLL was 2.3% (5 year incidence: 2.1%). Clinical characteristics (Rai stage III/IV) and biologic characteristics of the CLL B-cell (positive expression of ZAP-70, CD38 and CD49d; unmutated IGHV; and either del(11q22.3) or del(17p13) on FISH) at the time of diagnosis were associated with future risk of RS. Exposure to purine nucleoside analogues and/or alkylating agents for treatment of CLL was also associated with increased risk of RS. Disclosures: Zent: GlaxoSmithKline: Research Funding; Novartis: Research Funding; Genentech: Research Funding; Genzyme: Research Funding; Biothera: Research Funding. Shanafelt:Genentech: Research Funding; Glaxo-Smith-Kline: Research Funding; Cephalon: Research Funding; Hospira: Research Funding; Celgene: Research Funding; Polyphenon E International: Research Funding.

Abstract Background: Current drugs in myelofibrosis (MF), including JAK inhibitors, do not induce complete or partial remissions. Imetelstat is a 13-mer lipid-conjugated oligonucleotide that targets the RNA template of human telomerase reverse transcriptase. A phase-2 study in essential thrombocythemia showed platelet-lowering activity of imetelstat accompanied by reduction in JAK2V617F allele burden, thus providing the main rationale for conducting the current study. Methods: The current study (NCT01731951) is investigator-driven and IRB approved. Drug and research funding were provided by Geron Corporation (Menlo Park, CA, USA). The objective was to obtain preliminary information on therapeutic activity and safety of imetelstat in DIPSS-plus high or intermediate-2 risk MF. Adverse events (AEs) were monitored by Common Terminology Criteria for Adverse Events (Version 4.03) and responses by the International Working Group criteria (Blood2013). Laboratory correlative studies included measurement of plasma cytokines and analysis of mutations. Results : 33 patients were accrued and met eligibility criteria (median age 67 years; 67% males); type of MF was primary 55% and post-PV/ET 45%; risk distribution was high 48% and intermediate-2 52%. Karyotype was abnormal in 16 (48.5%) patients. 79% of patients had received prior therapy including JAK inhibitors in 33%. Protocol treatment : Imetelstat was administered by a 2-hour intravenous infusion (9.4 mg/kg) every three weeks (cohort A; n=19) or weekly x 3 followed by every three weeks (cohort B; n=16). As of 7/24/2014, 11 (33.3%) patients remain on active treatment (median number of cycles completed for all patients = 7; range 1-21). Three patients have died post-registration: intracranial hemorrhage (related), upper gastrointestinal hemorrhage (unrelated) and disease progression. Median follow-up for living patients was 11.0 months (range, 3.6 – 18.4). 22 (66%) patients have discontinued treatment, mainly because of suboptimal response (n=15) or disease progression (n=3). Toxicity : Treatment-related grade 4 neutropenia was seen in 6 (18%) patients and grade 4 thrombocytopenia in 7 (21%). Grade 3 anemia was seen in 9 (27%) patients. Two patients experienced grade 4+ non-hematologic AE: grade 5 intracranial hemorrhage (related), grade 5 upper gastrointestinal hemorrhage (not related); other grade 3+ non-hematologic AEs were seen in only one patient. Regardless of attribution, treatment-emergent grade 1 or 2 liver function test abnormalities affected bilirubin 46%, ALP 52%, AST 55% and ALT 24%. There were 3 instances of grade 3 ALP and one of bilirubin elevations. Efficacy : There were 7 (21.2%) complete (CR; n=4) or partial (PR; n=3) remissions, occurring at a median of 5 cycles of treatment (range 1-9). All 4 CR patients experienced reversal of BM fibrosis and 3 of them a complete molecular response. Six of the 7 CR/PR patients remain in remission (median 9.9 months, range 4.8-14.7). Other responses included anemia response in 4 (31%) of 13 transfusion-dependent patients, >50% reduction in palpable spleen size in 9 (39%) of 23 evaluable patients, ≥50% reduction in leukocyte count in 8 (80%) of 10 patients with marked leukocytosis (WBC >25 x 10(9)/L), resolution of leukoerythroblastosis in the majority of patients, and normalization of platelet count in 9 (75%) of 12 patients with thrombocytosis. Laboratory correlative studies : Mutational frequencies were 79% for JAK2, 18% for CALR, 3% for MPL, 33% for ASXL1, 9% for IDH1/2 and 33% for spliceosome component mutations. CR/PR rates were 27% in JAK2-mutated vs 0% unmutated (p=0.3) and 32% in ASXL1-unmutated vs 0% mutated (p=0.07). CR rate was 38% in SF3B1/U2AF1-mutated vs 4% unmutated (p=0.036). Grade 3+ neutropenia or thrombocytopenia was more likely to occur in JAK2-unmutated (p=0.02) and ASXL1-mutated cases (p=0.049). Multi-cytokine panel screening showed significant differences between baseline and post-treatment samples involving several cytokines, including IL-1b, IL-5, IL-7, IL-17, VEGF, IL-8 and TNF-α (p<0.001 for all). CR/PR did not correlate with baseline cytokine levels. Conclusions : The current study identifies imetelstat as an active drug in patients with MF, but also reveals its potential to cause significant myelosuppression. The association between CR/PR and specific mutations suggests potential targeted activity that might be exploited for patient and disease selection. Disclosures No relevant conflicts of interest to declare.

Abstract Abstract 2851 Background: It is widely appreciated that total cholesterol (TC) and triglyceride (TG) levels are decreased in patients with primary myelofibrosis (PMF). However, a systematic study looking into the serum lipid profile in PMF and its phenotypic and prognostic relevance is lacking. Methods: An updated Mayo Clinic database of 1008 karyotypically- and DIPSS-plus-annotated patients with PMF was used to identify study patients in whom serum lipid and lipoprotein levels were available at time of referral. Overall (OS) and leukemia-free (LFS) survivals were calculated from the time of referral, which was also the time of serum lipid analysis. Results: A total of 207 patients met the above stipulated criteria and had measurement of TC (n=207), TG (n=204), HDL cholesterol (HDL-C) (n=187), LDL cholesterol (LDL-C) (n=176) and non-HDL cholesterol (N-HDL-C) (n=184). The corresponding median (range) values (mg/dL) were 132 (57–296), 147 (42–742), 31 (8–96), 67 (11–186) and 104 (7–249). TC was significantly (P<0.05) lower in the presence of male sex, older age, red cell transfusion need, hemoglobin <10 g/dL, marked splenomegaly, circulating blasts, constitutional symptoms, JAK2 V617F, and ASXL1 mutations. TG was significantly (p<0.05) lower in the presence of JAK2 V617F, red cell transfusion need, hemoglobin <10 g/dL and thrombocytosis. DIPSS-plus risk categories showed significant differences in their TC (p<0.0001), HDL-C (p=0.004), LDL-C (p=0.004), and N-HDL-C (p=0.009) profiles with higher levels seen in lower risk patients; there were no correlations with TG levels. In univariate analysis, lower levels of TC, HDL-C, LDL-C and N-HDL-C were all associated with shortened survival (P<0.01 for each association) whereas survival was not affected by TG level (p=0.42). Multivariable analysis in the context of DIPSS-plus confirmed the independent prognostic relevance of both TC (p=0.05) and HDL-C (p=0.02) for OS. A similar analysis disclosed no correlation with LFS. Exclusion of patients receiving statin or other anti-lipid treatment did not affect the overall results. Patients in the lower quartiles for either TC (approximately <100 mg/dL) or HDL-C (approximately <25 mg/L) compared to those with higher levels displayed significantly shorter survival (Figure; RR 2.4, 95% CI 1.6–3.7; p<0.0001) that was not accounted for by DIPSS-plus (RR 1.6, 95% CI 1.1–2.5; p=0.03). Conclusions: Decreased levels of total or HDL cholesterol in PMF cluster with adverse disease features and carry a DIPSS-plus independent adverse prognostic value. Measurement of TC and HDL-C in PMF might be useful in monitoring disease progression and treatment response. Disclosures: No relevant conflicts of interest to declare.

Abstract Background Increased bone marrow (BM) reticulin fibrosis in polycythemia vera (PV) has been reported in 20% (Ann Hematol. 1999;78:495) to 51% (Eur J Haematol. 2011;86:148) of patients at diagnosis. In a previous report by the international working group for myeloproliferative neoplasms (MPN) research and treatment (IWG-MRT), the presence of BM fibrosis (≥ grade 1) at diagnosis was associated with a lower incidence of thrombosis during the clinical course and a higher risk of fibrotic progression while it did not affect overall (OS) or leukemia-free (LFS) survival (Blood. 2012;119:2239). The objectives for the current single center study of 262 PV patients were to validate the observations from the IWG-MRT and also identify other risk factors for myelofibrosis-free survival (MFFS) in PV. Methods Study patients were selected from our institutional database of MPN and fulfilled the 2016 World Health Organization (WHO) criteria for the diagnosis of PV (Blood. 2016;127:2391). Cytogenetic analysis and reporting was done according to the International System for Human Cytogenetic Nomenclature (Cytogenetic and Genome Research 2013;141:1-6). The degree of BM reticulin fibrosis was based on "real life" BM reports from Mayo Clinic hematopathologists and often in accordance with the European consensus scoring system (Haematologica 2005;90:1128). Statistical analyses considered clinical and laboratory parameters obtained at time of diagnosis. Results Patient characteristics: Analysis was conducted on 262 patients (median age 62 years; 50% males) who met 2016 WHO criteria for diagnosis of PV. Median (range) values were: hemoglobin 18 g/dl (14.8-24), leukocyte count 11.7 x109/L (4.3-59.3) and platelet count 454 x109/L (44-2747). Among informative cases, palpable splenomegaly was present in 27%, pruritus in 33% and erythromelalgia in 6%. Thrombosis history at diagnosis was documented in 28% of the patients and 23% experienced thrombotic events after diagnosis. Information on cytogenetics was available in 142 patients and karyotype was abnormal in 19%. BM reticulin fibrosis was reported to be absent in 135 patients (MF-0, 52%), grade 1 (MF-1) in 101 (39%), grade 2 (MF-2) in 22 (8%) and grade 3 (MF-3) in 4 (2%) patients. After a median follow up of 85 months, 107(41%) deaths, 30 (11%) fibrotic progression and 5 (2%) leukemic transformations were documented. Comparison of patients with and without bone marrow fibrosis A number of clinical and laboratory parameters were evaluated for possible association with the presence of ≥ grade 1 BM reticulin fibrosis and none, including age, sex, complete blood count, palpable splenomegaly, pruritus or erythromelalgia displayed a significant association. In univariate analysis, the presence of BM fibrosis (MF-0 versus MF-1 or greater) did not affect OS (p=0.5), LFS (p=0.2) or thrombosis-free survival (p=0.97) whereas a significant association was noted for MFFS (p=0.009; HR 2.9, 95% CI 1.3-6.7). Others risk factors for MFFS, in a univariate analysis, were leukocytosis ≥15 x 109/L (p=0.02; HR 2.7, 95% CI 1.17-6.48), presence of splenomegaly (p=0.02; HR 2.6, 95% CI 1.16-6) and abnormal karyotype (p=0.0005; HR 4.6, 95% CI 1.9-11). During multivariable analysis, not including karyotype, leukocytosis ≥15 x 109/L (p=0.04), presence of splenomegaly (p=0.04) and presence of BM reticulin fibrosis (p=0.01) remained significant; however, this significance for leukocytosis ≥15 x 109/L, presence of splenomegaly and BM reticulin fibrosis was lost when abnormal karyotype was added as covariate to each risk factor individually (p=0.4, p=0.1 and p=0.9, respectively). Conclusion We report a not infrequent (48% incidence) occurrence of ≥ grade 1 BM reticulin fibrosis at time of initial diagnosis of PV. In the current study, we did not find a prognostic impact for the presence of BM reticulin fibrosis, in terms of OS, LFS or thrombosis-free survival; however, a significant association was confirmed for MFFS that was independent of other non-genetic risk factors. The preliminary observation on the adverse prognostic impact of abnormal karyotype on MFFS requires additional studies. Disclosures No relevant conflicts of interest to declare.

Abstract Background : The 2016 revision to the World Health Organization (WHO) classification of myeloid neoplasms has recommended distinction between "proliferative" (WBC ≥ 13 x 10(9)/L) and "dysplastic" (WBC < 13 X 10(9)/L) subtypes of chronic myelomonocytic leukemia (CMML). In the current study of 261 molecularly-annotated cases, we sought to clarify the prognostic relevance of distinguishing proliferative from dysplastic CMML and also describe differences in the distribution of disease-associated mutations. Methods : 261 patients with WHO-defined CMML were included in the study. All patients had bone marrow (BM) biopsies and cytogenetics performed at diagnosis. Targeted capture assays were carried out on BM DNA specimens obtained at diagnosis for the following genes; TET2, DNMT3A, IDH1, IDH2, ASXL1, EZH2, SUZ12, SRSF2, SF3B1, ZRSR2, U2AF1, PTPN11, Tp53, SH2B3, RUNX1, CBL, NRAS, KRAS, JAK2, CSF3R, FLT3, KIT, CALR, MPL, NPM1, CEBPA, IKZF, and SETBP. The 2016 WHO criteria were used to sub-classify CMML into proliferative and dysplastic subtypes. Results :Among the 261 study patients, 65% were males and median age was 70 years. 154 (59%), 64 (25%) and 43 (16%) patients were classified as CMML-0, 1 and 2, respectively. At a median follow-up of 23 months, 174 (67%) deaths and 37 (14%) leukemic transformations were documented. Mutational frequencies were; TET2 45%, ASXL1 45%, SRSF2 40%, NRAS 14%, SETBP1 13%, CBL 10%, JAK2 7%, RUNX1 6%, U2AF1 6%, DNMT3A 6%, SF3B1 5%, ZRSR2 4%, Tp53 4%, IDH2 4%, KRAS 3%, PTPN11 2%, SH2B3 1%, CSF3R 1%, IDH1 1%, EZH2 1%, SUZ12 1%, KIT 1%, FLT3 1%, and CALR 1%. Risk stratification was based on the Mayo Molecular Model: 31% high, 30% intermediate-1, 28% intermediate-2 and 11 % low risk. i) Dysplastic versus proliferative CMML: phenotypic and molecular differences 139 (53%) patients had proliferative and 122 (47%) dysplastic subtypes. There was no difference between the CMML subtypes in terms of age and gender distribution, hemoglobin level, platelet count or BM blast content. Patients with proliferative CMML had higher absolute monocyte counts (AMC) (p<0.0001), circulating immature myeloid cells (IMC, p<0.001), circulating blasts (p<0.001) and serum LDH levels (p=0.01). The following gene mutations were more common in proliferative vs dysplastic CMML: ASXL1 (54% vs 37%, p=0.009), JAK2 (11% vs 3%, p=0.01) and CBL (11% vs 8%, p=0.047); SF3B1 mutations were more common in dysplastic CMML (8% vs 1%, p=0.02). There was no difference in the incidence of TET2, DNMT3A and SRSF2 mutations whereas there was a trend towards a higher prevalence of NRAS (p=0.06) and CSF3R (p=0.06) mutations in proliferative CMML. Cytogenetic abnormalities (p=0.03), including higher risk categories by the Spanish (p=0.03) and the Mayo-French (p=0.01) systems were more common in proliferative CMML. ii) Impact on overall and leukemia-free survival: Median survival for the entire cohort (n=261) was 24 months. In univariate analysis, survival was shorter in patients with proliferative (median 20 months) versus dysplastic (median 29 months) CMML (p=0.008; HR1.5, 95% CI 1.1-2.1; Figure 1A). Other variables of significance, in univariate analysis, included hemoglobin (p=0.001), leukocyte count (p=0.001), AMC (p=0.003), PB blast % (p=0.003), IMC (p=0.01), BM blast % (p=0.045), abnormal karyotype (p=0.02), ASXL1 (p=0.01) and DNMT3A (p=0.0003) mutations. In multivariable analysis, the difference in survival between proliferative and dysplastic subtypes remained significant with the addition of hemoglobin level (p=0.01), PB blast % (p=0.02), IMC (p=0.04), BM blast % (p=0.01) or DNMT3A mutations (p=0.01). This was, however, not the case with addition of leukocyte count (p=0.32), AMC (p=0.18) or ASXL1 mutational status (p=0.14); whereas the adverse impact on survival from the latter three parameters remained significant. The prognostic impact of ASXL1 mutations was most apparent in dysplastic CMML (Figure 1B). There was no difference in leukemic transformation rates (p=0.4). Conclusions: In the context of current prognostic models, sub-classification of CMML into proliferative and dysplastic subtypes might not provide additional prognostic value. The apparent difference in survival between the two subtypes of CMML is probably accounted for by the higher prevalence of leukocytosis/monocytosis and of ASXL1 mutations in proliferative CMML. Disclosures No relevant conflicts of interest to declare.

Abstract Background In a seminal report (NEJM 2011;30:2496), ASXL1, TP53, EZH2, ETV6 and RUNX1 mutations in myelodysplastic syndromes (MDS) were shown to carry an adverse prognostic impact that was independent of the international prognostic scoring system (IPSS) (Blood 1997;89:2079). Two recent studies (Leukemia20 May 2016; doi: 10.1038/leu.2016.138; Leukemia. 2014;28:241) have proposed mutation-enhanced prognostic models that include the revised IPSS (IPSS-R) (Blood 2012;120:2454 ). In the current study, we applied targeted next generation sequencing (NGS) in order to examine the prognostic interaction between adverse mutations and IPSS-R. Methods The study population was recruited from our institutional database of patients with primary MDS, based on availability of archived DNA from the time of diagnosis. The diagnosis of MDS and leukemic transformation (LT) was made according to WHO criteria (Blood. 2009;114:937). Targeted capture assays were carried out, on bone marrow DNA specimens, for the following genes: TET2, DNMT3A, IDH1, IDH2, ASXL1, EZH2, SUZ12, SRSF2, SF3B1, ZRSR2, U2AF1, PTPN11, Tp53, SH2B3, RUNX1, CBL, NRAS, JAK2, CSF3R, FLT3, KIT, CALR, MPL, NPM1, CEBPA, IKZF, and SETBP1.Specific variants were deemed as mutations if they are associated with a hematologic malignancy (as identified by COSMIC database), or if they have not been associated with a dbSNP. Results Patient characteristics and type and number of mutations: 179 patients were evaluated (median age 73 years; 68% males). IPSS-R risk distribution was 11% very high, 18% high, 16% intermediate, 39% low and 16% very low. At least one mutation was seen in 139 (78%) patients including 17% with one mutation, 29% with 2, 20% with 3, 10% with four, 2% with 5 and one patient with 6 mutations. The most frequent mutations were ASXL1 (30%), SF3B1 (20%), TET2 (17%), U2AF1 (16%) and SRSF2 (16%). Mutation clusters and clinical correlates: ASXL1 mutations were associated with the absence of SF3B1 (p=0.007), U2AF1 (p=0.01) and SRSF2 (p=0.003) mutations; SF3B1 with absence of U2AF1 (p=0.004) and SRSF2 (p=0.004) mutations; and U2AF1 with absence of SRSF2 mutations (p=0.01). Clinical correlates of ASXL1 mutations included older age, lower hemoglobin, and lower risk karyotype; SF3B1 with higher leukocyte count, higher platelet count, lower bone marrow blast percentage and lower risk karyotype; TET2 with older age and higher hemoglobin. Univariate overall and leukemia-free survival analysis before and after adjusting for IPSS-R: In univariate analysis, ASXL1, RUNX1 and TP53 mutations adversely and SF3B1 favorably affected survival; only ASXL1 mutations retained significance when analysis was adjusted for IPSS-R (HR 1.5, 95% CI 1.0-2.1; p=0.03). For leukemia-free survival, univariate analysis identified SRSF2, which was near-significant for overall survival (p=0.06), and RUNX1 mutations as adverse and SF3B1 mutations as favorable risk factors; SRSF2 (HR 4.1, 95% CI 1.6-10.2) and SF3B1-unmutated (HR 5.9, 95% CI 1.1-31.5) retained their significance when adjusted for IPSS-R. A borderline significance (p=0.07) indicating inferior survival for patients with three or more mutations was fully accounted for by the significant (P<0.0001) association between the presence of ASXL1 mutations and higher number of mutations. Multivariable analysis with individual IPSS-R variables: When the five IPSS-R variables (hemoglobin, platelets, absolute neutrophil count, bone marrow blast percentage and karyotype) were analyzed (both as continuous and IPSS-R-defined categorical variables) with each one of the aforementioned mutations with significant effect on overall or leukemia-free survival, only ASXL1 retained its significance for overall survival (HR 1.6, 95% CI 1.1-2.3) and SRSF2 (HR 5.2, 95% CI 2.1-13.3) for leukemia-free survival. Other IPSS-R variables that retained their significance for survival, in the presence of ASXL1 mutation status as a covariate, included hemoglobin level, platelet count and karyotype. Conclusions: In our cohort of 179 molecularly-annotated patients with newly-diagnosed primary MDS, ASXL1 and SRSF2 mutations were identified as IPSS-R-independent risk factors for overall and leukemia-free survival, respectively. The current study also suggests the need to re-evaluate currently established risk factors, in the context of prognostically-relevant molecular information. Disclosures No relevant conflicts of interest to declare.

Abstract Background: CD123 (alpha chain of the interleukin-3 receptor [IL-3RA]) heterodimerizes with CD131 (βc, common beta chain) to constitute the high-affinity receptor for IL-3. CD123 is not expressed on hematopoietic stem/progenitor cells from normal bone marrow (BM). In contrast, CD123 is highly expressed in CD34+/CD38- cells from acute myeloid leukemia (AML) patients, which recapitulate the leukemic phenotype in NOD/SCID mice; CD123, therefore, is a marker for leukemic stem cells in AML (Jordan CT et al., Leukemia 2000). CD123 is also highly expressed on CD4+/56+ leukemic cells in patients with blastic plasmacytoid dendritic cell neoplasm (BPDCN), an aggressive hematodermic neoplasm. The validity of CD123 as a rational therapeutic target was illustrated by clinical efficacy data for SL-401, a biologic target therapy directed to IL-3R, in patients with BPDCN and AML, respectively (Frankel AE et al., Blood 2014 andFrankel AE et al., J Clin Oncol 31, 2013 (suppl; abstr 7029)). Objectives: To study CD123 expression as a potential therapeutic target in myeloproliferative neoplasms (MPN). Methods: The study was approved by our institutional review board and patients provided written consent for sample collection. MPN diagnosis was based on WHO 2008 criteria. Surface antigen expression on hematopoietic cells of various lineages was interrogated using the 4-color multiparametric flow cytometer, FACSCaliber TM (BD Biosciences, San Jose, CA). Data analysis was performed using CellQuest Pro Software (BD Biosciences). Results: We studied a total of 21 MPN patients and 3 normal controls. Of the former, 14 patients had systemic mastocytosis (SM) (indolent SM=9, SM with associated myeloid neoplasm=4 and aggressive SM=1), 6 had primary myelofibrosis (PMF) and 1 had eosinophilic leukemia transformed to AML. Normal controls: Three normal BM samples were studied; the number of CD123 positive cells in the total population was <1%. Rare CD34+/CD38- cells were uniformly CD123-. Of the CD123+ cells, only a minor subset (<10%) coexpressed myeloid/granulocyte lineage (CD13+, CD15+ or CD16+) or monocyte/macrophage lineage (CD14+ or CD11b+) markers. Systemic mastocytosis: The data were informative for 6 patients (3 with peripheral blood [PB], 1 with BM, and 2 with paired PB and BM samples) for whom a sufficient number of mast cells (MC) were identified for immunophenotyping. Neoplastic MC were defined as CD117 hi/SSC hi/CD45 hi/CD34-/CD25+/FcεRI+. CD123 expression was seen in the majority of MC for 4 patients (#1-4, CD123 percentage positive, PB/BM): 91%/n.a. (#1), 75%/81% (#2), 84%/n.a. (#3), and 88%/n.a. (#4). In contrast, one patient (#5) had a minority of CD123+ MC (33%/28%). Clonal eosinophilia: PB was studied; the WBC differential count showed 41% leukemic blasts and 57% eosinophils (both confirmed to be clonally related based on FISH studies). Both cell populations were predominantly CD123+ (≥90%). Primary myelofibrosis: We studied PB samples from 6 PMF patients. Approximately 1-2% of circulating cells marked as CD123+; of these, expression was notable on CD34-/CD38+ cells (median 54%; range 41-74% positive). We found a larger proportion (30-50%) of CD123+ cells that coexpressed CD13+, CD16+ or CD11b+ representing monocytes, immature myeloid cells and granulocytes, as compared to normal BM controls. Studies on BM samples from PMF patients are ongoing. Conclusions: CD123 (IL-3RA) is expressed on relevant primary cells of interest in select MPNs, namely neoplastic mast cells in SM patients and eosinophils in clonal eosinophilia patients. In PMF, a minor population of circulating cells was CD123+ with a biased distribution on myeloid lineage cells as compared to normal BM samples. We are currently studying CD123 expression in additional MPN patients, and also analyzing CD123 expression on BM trephine biopsies by immunohistochemistry. In addition, the cytotoxicity assessment of SL-401 against MPN cell lines and primary cells is ongoing. Overall, the aforementioned data support the clinical development of SL-401 in patients with MPNs and clinical trials are currently being planned. Disclosures Brooks: Stemline Therapeutics: Employment, Equity Ownership. Pardanani:Stemline Therapeutics Inc.: Research Funding.

Abstract Abstract 2803 Background: Chronic myelomonocytic leukemia (CMML) is a clonal hematopoietic stem cell disorder with overlapping features between myelodysplastic syndromes (MDS) and myeloproliferative neoplasms. Ring sideroblasts (RS) represent abnormal mitochondrial iron accumulation in MDS; with ≥15% RS necessary for the conventional diagnosis of MDS-RS. Somatic spliceosome mutations are recurrent in MDS, with SF3B1 mutations being the most frequent in MDS-RS (∼75%) and SRSF2 in CMML (∼28%). The distribution of these mutations in the presence of both RS and monocytosis is unknown and their prognostic relevance, in the particular setting, undetermined. Methods: Using the Mayo Clinic database for myeloid malignancies (1997–2007), we identified patients who met the 2008 WHO criteria for CMML, and who also displayed at least 1% RS in their bone marrow (BM). All patients underwent BM examination and cytogenetic evaluation at diagnosis and the pathology slides, including iron stains, were centrally re-reviewed to accurately quantify BM RS. DNA was interrogated in the three most frequent spliceosome genes with somatic mutations; SF3B1, SRSF2 and U2AF35. Results: Sixty four patients met the above stipulated criteria for CMML with ≥1% RS; 46 (72%) were males and median age was 71 years (range, 17–90 years). Fifty three (83%) had CMML-1 and the remainder CMML-2. The percentage of patients with ≥15% RS was 41%: 30% had 15–49% RS and 11% had >50% RS. Thirty patients (47%) displayed SRSF2 mutations (mutational frequencies were 58% in the presence of <15% RS, 42% with 15–49% RS and 0% with >50% RS), 9 (14%) SF3B1 mutations (3% with <15% RS, 26% with 15–49% RS and 43% with >50% RS), and 5 (8%) U2AF35 mutations (8% with <15% RS, 11% with 15–49% RS and 0% with >50% RS). Mutational hotspots were P95 for SRSF2 (93%), K700 for SF3B1 (67%) and Q157 for U2AF35 (60%). The three spliceosome mutations were mutually exclusive. At a median follow-up of 26 months, 49 (77%) deaths and 11 (17%) leukemic transformations were documented. In univariate analysis, significant risk factors for survival included increased levels of white blood cell (WBC), absolute neutrophil (ANC), absolute monocyte (AMC), absolute lymphocyte (ALC) counts, the Spanish cytogenetics risk stratification system (Haematologica 2011;96:375), and the presence of circulating blasts. Neither the presence of spliceosome mutations (SF3B1/SRSF2/U2AF35) nor the percentage of RS (considered both as a continuous and a categorical variable), had an impact on either overall or leukemia-free survival. Conclusions: Among spliceosome mutations in CMML, those involving SRSF2 are by far the most frequent, even in the presence of ring sideroblasts. However, in patients with >50% RS, only SF3B1 mutations were seen whereas in those with 15–49% RS, SRSF2 mutations were more common. These observations suggest that SF3B1 mutations play a dominant but not exclusive role in the pathogenesis of RS. Regardless, the current study did not suggest prognostic impact from either the presence of the spliceosome mutations studied or the percentage of RS. Disclosures: No relevant conflicts of interest to declare.